PROJECT SUMMARY Mosquitoes transmit zika virus as well as other pathogens that cause many deadly diseases. These vectors cause more human suffering than any other group of animals and are responsible for millions of deaths worldwide. Insecticide sprays, use of repellents and insecticide-treated bed nets for adult control and use of larvicides are the most common methods used to control disease vectors. However, widespread resistance development to insecticides in mosquitoes, human health and environmental concern from the residual toxicity of these chemicals are limiting the use of these strategies. Therefore, there is an urgent need to develop novel strategies to control disease vectors. Many applications of RNA interference (RNAi) are being developed for use in human health. RNAi could also help with studies in vector biology as well as in developing advanced vector control methods. RNAi efficiency is variable among insects; works very well in coleopteran insects but not in insects belonging to order Diptera that includes mosquitoes. One of the main challenges in the widespread use of RNAi technology in disease vectors is the lack of efficient and reliable dsRNA delivery method. We propose to develop novel formulations of double-stranded RNA (dsRNA) composited with polymers and/or particulate cores resulting in nanostructured delivery vehicles, or dsRNA nanocomposites (dsRNA-NCs). The major goal of these studies is to improve RNAi efficiency in mosquitoes that transmit Zika virus for applications in basic research as well as in the development of vector control methods. In specific Aim 1, we will prepare dsRNA-NCs and determine their properties. We hypothesize that biodegradable and environmentally friendly biomaterials could be used to develop prototype dsRNA-NCs that could aid in enhancing RNAi efficiency in mosquitoes. We will prepare cores of Ca3(PO4)2 coated with chitosan, Poly-L-Lysine or Poly- L-histidine. Additionally, Au cores will be used as an insoluble probe. A number of analytical methods will be used to determine the properties of synthesized dsRNA-NCs. We will also perform a comparative analysis of synthesized dsRNA- NCs by studying protection of dsRNA from dsRNases, its uptake, tissue distribution, intracellular transport including endosomal escape and dsRNA processing in Aeg-2 cells (a cell line developed from Aedes aegypti), Ae. aegypti larvae and adults as a model vector species. In Specific Aim 2, we will determine the efficacy and non-target effects of synthesized dsRNA-NCs in Ae. aegypti larvae and adults. We will expose Aeg-2 cells or feed Ae. aegypti larvae and adults with dsRNA- NCs and determine their efficiency in knocking down target genes and causing mortality as well as their effect on the expression of non-target genes. This application proposes to employ novel approaches, including nanotechnology to improve RNAi for use in disease vectors. The proposal uses interdisciplinary approaches to exploit modern technology for improvement in the health of citizens. Successful completion of proposed research will provide novel tools to study the biology of vectors of Zika and other pathogens and develop modern vector control methods.